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L M Nadler

Publications and source records attributed to L M Nadler.

At least 19 recordsLinked to original sources
PubMed1992

Signalling through the MHC class II cytoplasmic domain is required for antigen presentation and induces B7 expression.

Class II major histocompatibility complex (MHC) molecules function as antigen-presenting elements as well as signal transducers on B lymphocytes. We previously reported that a B lymphoma cell transfectant, 5C2, expressing genetically engineered I-Ak molecules with truncated cytoplasmic domains was severely impaired in both antigen presentation and in anti-Ia-induced intracytoplasmic signalling. These two functions could be restored by preculturing 5C2 cells with cyclic AMP analogues. Here we demonstrate that impaired signal transduction by truncated class II molecules results in a deficiency in induction of the newly defined B-cell accessory molecule B7 (ref. 8), which can be reversed by restoration of B7 expression. These data imply that contact of the T-cell antigen receptor with MHC/antigen ligand results in signal transmission through the class II cytoplasmic domain. This signal, which can be mimicked by dibutyryl cAMP, induces expression of B7, resulting in effective antigen presentation. The fact that crosslinking of surface class II MHC also induces B7 expression on normal resting human B cells supports this contention.

Animals↗
PubMed1992

Follicular dendritic cells inhibit human B-lymphocyte proliferation.

In germinal centers, B lymphocytes are intimately associated with follicular dendritic cells (FDCs). It has been hypothesized that FDCs are involved in the regulation of B-cell growth and differentiation through cell-cell interactions. In this study, highly enriched preparations of FDCs were isolated by cell sorting using the FDC restricted monoclonal antibody DRC-1. When irradiated FDCs were cultured with mitogen stimulated B cells, B cell 3H-TdR uptake was inhibited by up to 80%. This inhibitory effect was not seen when paraformaldehyde fixed FDCs were added to B-cell cultures, suggesting that the FDCs needed to be metabolically active. Moreover, supernatants from cultured FDCs were similarly able to inhibit B-cell proliferation. These results demonstrate that FDCs may downregulate the clonal expansion of B cells that occurs within lymphoid follicles as part of the normal physiologic immune response. Potentially, the loss of the inhibitory role of FDCs in vivo may be of importance in certain infectious and neoplastic processes in which germinal centers are affected.

B-Lymphocytes↗
PubMed1992

Bone marrows of non-Hodgkin's lymphoma patients with a bcl-2 translocation can be purged of polymerase chain reaction-detectable lymphoma cells using monoclonal antibodies and immunomagnetic bead depletion.

Using the extremely sensitive technique of polymerase chain reaction (PCR) to detect the bcl-2 translocation, only 50% of bone marrows could be purged of PCR-detectable lymphoma cells using a cocktail of three anti-B-cell monoclonal antibodies (MoAbs) and complement-mediated lysis. This observation is of clinical importance because those patients whose reinfused marrows harbored residual lymphoma cells showed a significantly increased incidence of relapse. To improve purging, we used PCR detection of the bcl-2 translocation to compare the efficiency of complement-mediated lysis with immunomagnetic bead depletion. Using either a three or a four MoAb cocktail followed by immunomagnetic bead depletion, all PCR-detectable cells were purged after three cycles of treatment. In these same patient samples, treatment with three MoAbs and complement purged only 11 of the 25 (44%) samples. The addition of a fourth MoAb followed by complement lysis purged the marrows of only an additional five patients. Immunomagnetic bead depletion was specific because there was no loss of committed myeloid progenitor cells. The above results suggest that immunomagnetic bead depletion of the harvested marrow will likely be superior to our previous method of purging and the lack of nonspecific toxicity to myeloid progenitor cells predicts that it will not impair engraftment. This methodology will now be used to determine whether the reinfusion of lymphoma free marrow affects the incidence of relapse after autologous bone marrow transplantation.

Antibodies, Monoclonal↗
PubMed1992

Correlation between in vivo toxicity and preclinical in vitro parameters for the immunotoxin anti-B4-blocked ricin.

Anti-B4-blocked ricin (Anti-B4-bR) is an immunotoxin comprised of the anti-B4 monoclonal antibody and the protein toxin, "blocked ricin." In blocked ricin, the galactose-binding sites of the ricin B-chain which mediate nonspecific binding to cells are blocked by covalently linked affinity ligands prepared from N-linked oligosaccharides of fetuin. Blocked ricin consists of two species, one with two covalently attached ligands and one with three covalently attached ligands. In a Phase I dose escalation clinical trial, Anti-B4-bR was administered to patients with relapsed and refractory B-cell neoplasms by 7-day continuous infusion. Although several different lots of Anti-B4-bR had similar IC37 values as determined by in vitro cytotoxicity testing on cultured human cell lines, these lots differed in their in vivo toxicity when administered to patients. Thus, IC37 values alone were not sufficient to predict in vivo toxicity. We report that the degree of cell kill at concentrations of drug that saturate the B4 antigen and murine 50% lethal dose values provide additional parameters that may be predictive of in vivo cytotoxicity. Furthermore, we performed detailed cytotoxicity studies of the ricin species containing two and three covalently attached ligands, respectively. In vitro cytotoxicity testing using these samples revealed that Anti-B4-bR made with blocked ricin containing two covalently attached ligands is capable of depleting five logs of target cells in an in vitro cytotoxicity assay, while Anti-B4-bR comprised of blocked ricin with three ligands can deplete only one log of cells. Log cell kill at antigen saturating concentration, murine 50% lethal dose and biochemical analysis of the composition of blocked ricin are therefore important considerations for establishing the potential efficacy and safety of Anti-B4-bR.

Alanine Transaminase↗
PubMed1992

Early signaling defects in human T cells anergized by T cell presentation of autoantigen.

Major histocompatibility complex class II-positive human T cell clones are nontraditional antigen-presenting cells (APCs) that are able to simultaneously present and respond to peptide or degraded antigen, but are unable to process intact protein. Although T cell presentation of peptide antigen resulted in a primary proliferative response, T cells that had been previously stimulated by T cells presenting antigen were completely unresponsive to antigen but not to interleukin 2 (IL-2). In contrast, peptide antigen presented by B cells or DR2+ L cell transfectants resulted in T cell activation and responsiveness to restimulation. The anergy induced by T cell presentation of peptide could not be prevented by the addition of either autologous or allogeneic B cells or B7+ DR2+ L cell transfectants, suggesting that the induction of anergy could occur in the presence of costimulation. T cell anergy was induced within 24 h of T cell presentation of antigen and was long lasting. Anergized T cells expressed normal levels of T cell receptor/CD3 but were defective in their ability to release [Ca2+]i to both alpha CD3 and APCs. Moreover, anergized T cells did not proliferate to alpha CD2 monoclonal antibodies or alpha CD3 plus phorbol myristate acetate (PMA), nor did they synthesize IL-2, IL-4, or interferon gamma mRNA in response to either peptide or peptide plus PMA. In contrast, ionomycin plus PMA induced both normal proliferative responses and synthesis of cytokine mRNA, suggesting that the signaling defect in anergized cells occurs before protein kinase C activation and [Ca2+]i release.

Antigen-Presenting Cells↗
PubMed1992

Human bone marrow depleted of CD33-positive cells mediates delayed but durable reconstitution of hematopoiesis: clinical trial of MY9 monoclonal antibody-purged autografts for the treatment of acute myeloid leukemia.

The CD33 antigen, identified by murine monoclonal antibody anti-MY9, is expressed by clonogenic leukemic cells from almost all patients with acute myeloid leukemia; it is also expressed by normal myeloid progenitor cells. Twelve consecutive patients with de novo acute myeloid leukemia received myeloablative therapy followed by infusion of autologous marrow previously treated in vitro with anti-MY9 and complement. Anti-MY9 and complement treatment eliminated virtually all committed myeloid progenitors (colony-forming unit granulocyte-macrophage) from the autografts. Nevertheless, in the absence of early relapse of leukemia, all patients showed durable trilineage engraftment. The median interval post bone marrow transplantation (BMT) required to achieve an absolute neutrophil count greater than 500/microL was 43 days (range, 16 to 75), to achieve a platelet count greater than 20,000/microL without transfusion was 92 days (range, 35 to 679), and to achieve red blood cell transfusion independence was 105 days (range, 37 to 670). At the time of BM harvest, 10 patients were in second remission, one patient was in first remission, and one patient was in third remission. Eight patients relapsed 3 to 18 months after BMT. Four patients transplanted in second remission remain disease-free 34+, 37+, 52+, and 57+ months after BMT. There was no treatment-related mortality. Early engraftment was significantly delayed in patients receiving CD33-purged autografts compared with concurrently treated patients receiving CD9/CD10-purged autografts for acute lymphoblastic leukemia or patients receiving CD6-purged allografts from HLA-compatible sibling donors. In contrast, both groups of autograft patients required a significantly longer time to achieve neutrophil counts greater than 500/microL and greater than 1,000/microL than did patients receiving normal allogeneic marrow. CD33(+)-committed myeloid progenitor cells thus appear to play an important role in the early phase of hematopoietic reconstitution after BMT. However, our results also show that human marrow depleted of CD33+ cells can sustain durable engraftment after myeloablative therapy, and provide further evidence that the CD33 antigen is absent from the human pluripotent hematopoietic stem cell.

Adult↗
PubMed1992

Antibody and B7/BB1-mediated ligation of the CD28 receptor induces tyrosine phosphorylation in human T cells.

CD28 is an adhesion receptor expressed as a 44-kD dimer on the surface of a major subset of human T cells. The CD28 receptor regulates the production of multiple lymphokines, including interleukin 2 (IL-2), by activation of a signal transduction pathway that is poorly understood. Here we show that ligation of CD28 by a monoclonal antibody (mAb) or by a natural ligand, B7/BB1, induces protein tyrosine phosphorylation that is distinct from T cell receptor (TCR)-induced tyrosine phosphorylation. CD28-induced protein tyrosine phosphorylation was greatly enhanced in cells that had been preactivated by ligation of the TCR, or by pretreatment with phorbol esters. Rapid and prolonged tyrosine phosphorylation of a single substrate, pp100, was induced in T cells after interaction with B7/BB1 presented on transfected Chinese hamster ovary (CHO) cells. Anti-B7 mAb inhibited B7/BB1 receptor-induced tyrosine phosphorylation, indicating that B7-CD28 interaction was required. CD28-induced tyrosine phosphorylation was independent of the TCR because it occurred in a variant of the Jurkat T cell line that does not express the TCR. Herbimycin A, a protein tyrosine kinase inhibitor, could prevent CD28-induced tyrosine phosphorylation and CD28-induced IL-2 production in normal T cells. The simultaneous crosslinking of CD28 and CD45, a tyrosine phosphatase, could prevent tyrosine phosphorylation of pp100. These results suggest that specific tyrosine phosphorylation, particularly of pp100, occurs directly as a result of CD28 ligand binding and is involved in transducing the signal delivered through CD28 by accessory cells that express the B7/BB1 receptor. Thus, this particular form of signal transduction may be relevant to lymphokine production and, potentially may provide a means to study the induction of self-tolerance, given the putative role of the costimulatory signal in the induction of T cell activation or anergy.

Antibodies, Monoclonal↗
PubMed1992

Growth arrest of human B lymphocytes is accompanied by induction of the low molecular weight mammalian heat shock protein (Hsp28).

A large number of protein and molecular markers have been identified that delineate the early stages of human B cell activation and proliferation. In contrast, few if any molecules are transiently expressed precisely as activated B cells stop proliferating and undergo growth arrest. We demonstrate that the low molecular weight heat shock protein (hsp28) exhibits unique induction kinetics that specifically demarcates this interval. After mitogenic activation of unstimulated splenic B cells, hsp28 protein and phosphorylation transiently increase coinciding precisely with the peak of cellular proliferation and the onset of growth arrest. Although most neoplastic B cells constitutively express hsp28, three cell lines were identified that were hsp28-. No differences in phenotype or growth kinetics were detected between hsp28+ and hsp28- neoplastic B cells demonstrating that hsp28 expression is not essential for cell growth. However, when treated with phorbol ester or heat shock, these hsp28- cell lines synthesize hsp28 followed by the onset growth arrest. The consistency with which hsp28 induction transiently delineates the interval from peak proliferation to the onset of growth arrest suggests hsp28 itself is likely to be involved in regulating this process.

B-Lymphocytes↗
PubMed1992

The gene for B7, a costimulatory signal for T-cell activation, maps to chromosomal region 3q13.3-3q21.

B7 is an activation antigen expressed on activated B cells and gamma-interferon-stimulated monocytes. The B7 antigen is the natural ligand for CD28 on T cells. After engagement of T-cell receptor with antigen in association with major histocompatibility complex class II, a second signal mediated through the binding of B7 to CD28 greatly upregulates the production of multiple lymphokines. We have now mapped the B7 gene to human chromosome 3 using the technique of polymerase chain reaction on a panel of hamster x human somatic cell hybrid DNAs. We have further localized the gene to 3q13.3-3q21 using in situ hybridization on human metaphase chromosomes. Trisomy of chromosome 3 is a recurrent chromosome change seen in various lymphomas and lymphoproliferative diseases, particularly diffuse, mixed, small, and large cell lymphomas, human T-cell lymphotropic virus type I-induced adult T-cell leukemia, and angioimmunoblastic lymphadenopathy. A number of chromosomal defects involving 3q21 have been described in acute myeloid leukemia and also in myelodysplastic and myeloproliferative syndromes. The mapping of B7 may permit further insight into disease states associated with aberrant lymphocyte activation and lymphokine synthesis.

Animals↗
PubMed1992

Murine B7 antigen provides an efficient costimulatory signal for activation of murine T lymphocytes via the T-cell receptor/CD3 complex.

We demonstrate that the murine B7 (mB7) protein is a potent costimulatory molecule for the activation of resting murine CD4+ T cells through the T-cell receptor (TCR)/CD3 complex. Stable mB7-transfected Chinese hamster ovary cells, but not vector-transfected controls, synergize with anti-CD3 monoclonal antibody and Con A-induced T-cell activation, resulting ultimately in proliferation. mB7 exerted its effect by inducing production of interleukin 2 and expression of the interleukin 2 receptor. Thus, mB7 costimulates T-cell activation through the TCR/CD3 complex by positively modulating the normal pathway of T-cell expansion. In contrast to the pronounced effect of mB7 on the activation of T cells through the TCR/CD3 complex, the mB7-transfected CHO cell line costimulated T-cell activation via the glycosylphosphatidylinositol-anchored proteins Thy-1 and Ly-6A.2 only inefficiently. Finally, the combination of a calcium ionophore and mB7 is not sufficient to cause T-cell proliferation, while the combination of a calcium ionophore and phorbol 12-myristate 13-acetate (PMA) stimulates T cells efficiently. The signals that mB7 and PMA provide for murine T lymphocyte activation are therefore not interchangeable, although both costimulate activation through the TCR/CD3 complex.

Animals↗
PubMed1992

Follicular non-Hodgkin's lymphoma cell adhesion to normal germinal centers and neoplastic follicles involves very late antigen-4 and vascular cell adhesion molecule-1.

Follicular lymphomas recapitulate the architecture of germinal centers (GCs) of normal secondary lymphoid follicles. Using an in vitro binding assay, it has recently been demonstrated that the normal B lymphocytes bind to GCs. This interaction is mediated by a receptor-ligand pair consisting of the beta 1 integrin very late antigen 4 (VLA-4) on the B cell, and the vascular cell adhesion molecule-1 (VCAM-1) expressed on follicular dendritic cells (FDC). Considering the similarities between follicular lymphomas and normal GCs, the adhesive interaction of follicular non-Hodgkin's lymphoma (NHL) cells and GCs was examined. Cells isolated from 16 of 24 cases of follicular NHL bound to normal GCs. Neoplastic follicles could similarly support the binding of follicular NHL cells. This adhesion was inhibited by monoclonal antibodies (MoAbs) directed against VLA-4 and VCAM-1. This supports the hypothesis that the neoplastic follicles used the identical adhesive interactions responsible, at least in part, for the localization of normal B cells to GCs. Adhesion receptors have an important role in the regulation of normal lymphoid cell proliferation, differentiation, and localization. Therefore, an understanding of the adhesive interaction of follicular NHL cells with GCs may provide insight into the clinical and biologic behavior of these diseases.

Antibodies, Monoclonal↗
PubMed1991

All advanced stage non-Hodgkin's lymphomas with a polymerase chain reaction amplifiable breakpoint of bcl-2 have residual cells containing the bcl-2 rearrangement at evaluation and after treatment.

Polymerase chain reaction (PCR) of bcl-2 provides an extremely sensitive method to detect minimal disease in approximately 50% of patients with non-Hodgkin's lymphomas (NHL). In an attempt to determine the clinical usefulness of this technique, we examined the bone marrow (BM) of 152 patients with advanced-stage NHL at the time of evaluation and after induction or salvage chemotherapy before autologous BM transplantation. The BM proved to be an accessible and reproducible tissue source to determine PCR positivity because all of the 102 patients examined had the same PCR-amplifiable breakpoint in their BM and lymph node. At the time of evaluation, PCR analysis in advanced-stage NHL patients added little additional information to morphologic analysis because each technique identified BM infiltration in approximately 70% of patients. PCR was significantly more useful in determining BM infiltration after induction or salvage therapy. At that time, approximately 50% of patients had morphologically normal BM, whereas PCR analysis remained positive in 100% of those with an amplifiable breakpoint. These observations were confirmed in a clinical trial attempting to induce remission in previously untreated low-grade advanced-stage NHL patients. In this series, PCR was positive in all patients after treatment although the BM was histologically uninvolved in 50% of cases, showing that conventional therapy did not eradicate bcl-2-positive cells.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1991

An immunotoxin prepared with blocked ricin: a natural plant toxin adapted for therapeutic use.

Ricin, the cytotoxic protein isolated from castor beans, is composed of two subunits, A-chain and B-chain. Ricin intoxicates cells by binding through its B-chain to galactose-terminated oligosaccharides found on the surface of all eukaryotic cells and then transferring its A-chain to the cytosol where it disrupts protein synthesis by inactivating ribosomes. In addition to binding, the B-chain plays an important, but not yet understood, role in the translocation of the A-chain through a cellular membrane to the cytosol. Blocking the two galactose-binding sites of native ricin by chemical modification with affinity ligands created an altered toxin, called blocked ricin, that has at least a 3500-fold lower binding affinity and is more than 1000-fold less cytotoxic than native ricin for Namalwa cells (a Burkitt's lymphoma line) but that has maintained the translocation function of the B-chain and the catalytic activity of the A-chain. Conjugation of blocked ricin to monoclonal antibodies that bind to cell surface antigens creates new cytotoxins that approach the potency of native ricin. These cytotoxins incorporate the three essential functions of natural toxins, i.e., binding to cells, transport through a membrane, and catalytic inactivation of an essential cellular process; but in addition they possess a defined cellular target specificity. Such potent immunotoxins may play an important therapeutic role in cancer treatment. Clinical trials with an anti-CD19-blocked ricin and an anti-CD33-blocked ricin conjugate against B-cell cancers and acute myeloblastic leukemia have begun.

Animals↗
PubMed1991

Selective induction of B7/BB-1 on interferon-gamma stimulated monocytes: a potential mechanism for amplification of T cell activation through the CD28 pathway.

The B cell activation antigen B7/BB-1 is the natural ligand for the T cell antigen CD28 and these two molecules are capable of mediating T-B cell adhesion. Engagement of the CD28 pathway provides a costimulatory signal to T cells leading to enhanced lymphokine production. We report that interferon-gamma (INF-gamma) induces the expression of B7/BB-1 on monocytes. This induction was very specific since other cytokines and stimuli which activate monocytes including M-CSF, GM-CSF, IL3, TNF-alpha, and LPS were unable to induce B7/BB-1. Following culture of monocytes with INF-gamma, maximal mRNA and cell surface B7/BB-1 expression was detected at 12 and 24 hr, respectively. In addition to antigen presentation, optimal T cell activation and lymphokine synthesis require an additional cell to cell contact signal provided by the antigen presenting cell. The induction of B7/BB-1 on monocytes and subsequent heterophilic interaction of B7/BB-1 with CD28 may provide a mechanism for the amplification of T cell proliferation and lymphokine production by INF-gamma activated monocytes.

Antigens, CD↗
PubMed1991

Structure, expression, and T cell costimulatory activity of the murine homologue of the human B lymphocyte activation antigen B7.

Following occupancy of the T cell receptor by antigen, T cell proliferation and lymphokine production are determined by a second costimulatory signal delivered by a ligand expressed on antigen presenting cells. The human B cell activation antigen B7, which is expressed on antigen presenting cells including activated B cells and gamma interferon treated monocytes, has been shown to deliver such a costimulatory signal upon attachment to its ligand on T cells, CD28. We have cloned and sequenced the murine homologue of the human B7 gene. The predicted murine protein has 44% amino acid identity with human B7. The greatest similarity is in the Ig-V and Ig-C like domains. Murine B7 mRNA was detected in murine hematopoietic cells of B cell but not T cell origin. Cells transfected with murine B7 provided a costimulatory signal to human CD28+ T lymphocytes. These results demonstrate the costimulatory activity of murine B7 and provide evidence that the ligand attachment site is conserved between the two species.

Amino Acid Sequence↗